Composition of matter and process

ABSTRACT

Disclosed are degradation products of the antibiotics rubradirin and rubradirin B and processes for their preparation. Some of these products have antibacterial activity, and, thus, can be used in various environments to inhibit susceptible bacteria. Also, some of these products can be used as intermediates to make useful antibacterials.

CROSS REFERENCE TO RELATED APPLICATION

This is a division of application Ser. No. 874,767, filed Feb. 6, 1978,now U.S. Pat. No. 4,137,410, issued Jan. 30, 1979 which is acontinuation-in-part of application Ser. No. 793,785, filed May, 5,1977, now abandoned.

BACKGROUND OF THE INVENTION

The antibiotic rubradirin, and a microbiological process for preparingthe same, are disclosed in U.S. Pat. No. 3,335,057. The structure ofrubradirin has been tentatively determined to be as follows: ##STR1##

The structure of rubradirin B has been tentatively determined to be asfollows: ##STR2##

BRIEF SUMMARY OF THE INVENTION

Upon subjecting rubradirin to base hydrolysis there are obtained novelcompounds which can be shown as follows: ##STR3##

If rubradirin is reacted with an amine, e.g. aqueous methylamine, theamide of IV is obtained.

Acid hydrolysis of rubradirin gives novel compounds which can be shownas follows: ##STR4##

Upon reacting compound V with a saturated solution of ammonia andmethanol, there are obtained compound III and also another novelcompound which can be shown as follows: ##STR5##

If compound V is reacted with primary amines such as ethylamine, theethylamide corresponding to VII is obtained.

Degradation of rubradirin in acetic anhydride and pyridine yields anovel compound which can be shown as follows: ##STR6##

A similar compound having the structure: ##STR7## wherein Ac is acetyl,is known in the prior art. See U.S. Pat. No. 3,105,088.

Base hydrolysis of rubradirin B gives novel compounds which can be shownas follows: ##STR8##

DETAILED DESCRIPTION OF THE INVENTION

Upon subjecting rubradirin to base hydrolysis there are obtainedcompounds III and IV. The reaction can be conducted with ammoniacal,alkali or alkaline earth metal bases, for example, ammonium hydroxide,sodium hydroxide (preferred), potassium hydroxide, and calciumhydroxide. The pH of the reaction, advantageously, is maintained in therange of 10 to about 12.5 for about 36-60 hours, while the temperatureis about 20° to about 30° C. The resulting degradation products can berecovered from the reaction mixture by first acidifying the mixture to apH of about 2.5 with a mineral acid; hydrochloric acid is preferred. Thewater-insoluble fraction can be extracted with a suitable solvent, forexample, chloroform (preferred) or ethyl acetate. The desired productscan be precipitated from the solvent extract with Skellysolve B(isomeric hexanes). This material then can be subjected tochromatographic procedures on silica gel to yield compounds III and IVas separate entities. Compound III is active against the bacteriumSarcina lutea on a standard agar disc plate assay (80 BU/mg). A biounit(BU) is defined as the concentration of the antibiotic which gives a 20mm zone of inhibition when 0.08 ml of antibiotic solution is applied toa 12.5 mm diameter adsorbent paper disc. Compound III can be placed intosolution for the assay by dissolving in methanol, or acetone, ordimethylformamide and diluting with phosphate buffer at pH 7.8.

Since compound III is active against S. lutea, it can be used to swablaboratory benches and equipment in a bacteriology laboratorycontaminated with S. lutea. Further, it can be used to treat opencooling water systems in which S. lutea has been found to be one of thecontaminants.

Compounds III and IV can be used as ultraviolet screeners in theplastics industry since they effectively absorb UV rays. For example,they can be used in polyolefin resin formulations for numerous plasticapplications to prevent degradation by sunlight.

Acid hydrolysis of rubradirin gives novel compounds V and VI. Thehydrolysis can be conducted using acetic acid (preferred) or a mineralacid in about 80% methanol or ethanol. The reaction, advantageously, ismaintained at a pH of about 2 to 3 and at a temperature of about 20° toabout 30° C. for about 6 days. Filtration of the reaction mixtureaffords a red crystalline precipitate which is active against S. luteaand is identified as compound V. Compound VI can be isolated from theremaining filtrate by first evaporating the filtrate to a residue andthen dissolving the residue in chloroform. The chloroform solution,advantageously, can be decolorized by extraction with a 3% aqueoussodium carbonate solution. The dried chloroform solution can be dilutedby addition of methanol, followed by evaporation to yield white crystalsof compound VI.

Since compound V is active against S. lutea (150 BU/mg), it can be usedfor the same purposes as described above for compound III. It can alsobe used as an ultraviolet screener, as described above for compounds IIIand IV.

Compound VI is a stereoisomer of the compound disclosed in JACS, VOl.90, page 7129 (1968). Compound VI can be used as an intermediate for thepreparation of the corresponding amino sugar according to the proceduresdisclosed in U.S. Pat. No. 3,996,205. Further, the nitro group incompound VI can be reduced to an amine using standard catalyticreduction procedures with Raney nickel or 10% palladium on charcoal.This amine compound can then be used to make antibacterially activeantibiotics in accord with the procedures disclosed in U.S. Pat. No.3,996,205.

Upon reacting compound V with a saturated solution of ammonia andmethanol for about 16 hours at about 20° to about 30° C., there areproduced compounds III and VII. The reaction mixture can be evaporatedto dryness and the residue triturated with a two-phase mixture ofchloroform and 0.1 N sulfuric acid. This reaction mixture then can befiltered to give a precipitate containing compound VII. The chloroformphase of the filtrate can be washed, evaporated and dried to yieldcompound III. Compound VII can be purified from the first precipitate bycrystallization from hot methanol.

Compound VII can be used as an ultraviolet screener in the plasticsindustry, as disclosed above, since it effectively absorbs UV rays.

A mixture of rubradirin, pyridine, and acetic anhydride when refluxedfor about 2 to about 4 hours yields compound VIII. This compound can berecovered from the reaction mixture by first pouring the mixture on iceto give a precipitate which can be removed by filtration andrecrystallized from methanol twice to give white crystals of compoundVIII. Compound VIII also can be used as an ultraviolet screener in theplastics industry since it effectively absorbs UV rays.

Base hydrolysis of rubradirin B yields compounds X and XI. The reactioncan be conducted with ammoniacal, alkali or alkaline earth metal bases,for example, ammonium hydroxide, sodium hydroxide (preferred), potassiumhydroxide, and calcium hydroxide. The pH of the reaction,advantageously, is maintained in the range of 10 to about 12.5 for about2 to 3 days. The temperature during this time is maintained at about 20°to about 30° C., preferably at 22° C. These compounds can be recoveredfrom the reaction mixture by first acidifying the mixture to about pH 2with a mineral acid; sulfuric acid is preferred. The insoluble andchloroform-soluble fractions are pooled. This material can bechromatographed on silica gel 60 to give compound X by elution withchloroform and methanol mixtures. Compound XI can then be isolated fromthe chromatographic column by elution with methanol.

Compound X is active against S. lutea (6 BU/m) and, thus, can be used toinhibit this bacterium in the same manner as disclosed above forcompound III. Further, compound X can be used as an ultraviolet screenerin the plastics industry since it effectively absorbs UV rays. Likewise,compound XI can be used as an ultraviolet screener in the plasticsindustry.

The following examples are illustrative of the process and products ofthe present invention, but are not to be construed as limiting. Allpercentages are by weight, and solvent mixture proportions are by volumeunless otherwise noted.

EXAMPLE 1--Base Degradation Of Rubradirin: Preparation Of Compound IIIAnd Compound IV

A solution containing 1 g. (1 mmol) of rubradirin, 80 mg (2 mmol) ofsodium hydroxide and 50 ml of water is stored at room temperature for 42hours. The pH is 11.5. It is then acidified to pH 2.5 with about 2 ml of2 N hydrochloric acid and the water-insoluble fraction is taken intochloroform, washed with water and precipitated with Skellysolve B,affording 850 mg of red powder.

This is then chromatographed on pH 5.8-buffered silica gel developedfirst with chloroform. The first red band, when isolated, consists of125 mg of unchanged rubradirin. The second red band is found to contain160 mg of a red powder; base degradation product compound III. Anal.Calcd. for C₂₃ H₂₃ NO₈ : N, 3.28; MW 441.1424. Found: N, 3.17; MW441.1417. Compound III assays 80 BU/mg on a standard disc plate S. luteaassay.

The column is then leached with chloroform:methanol (99:1 v/v) whichremoves some color but minimal solids. A final development withchloroform:methanol (99:5 v/v) removes a brown band which contains 600mg of light tan crystals; base degradation compound IV. Anal. Calcd. forC₂₅ H₂₅ N₃ O₁₃.

EXAMPLE 2--Acid Degradation Of Rubradirin: Preparation Of Compound V AndCompound VI

A. A 2 g quantity of rubradirin in 300 ml of glacial acetic acid isdiluted with 100 ml of water and the mixture is stirred at roomtemperature for 6 days. Filtration affords 1.08 g of compound V, a redcrystalline precipitate. This material assays 140 BU/mg (S. lutea).

B. To 3.4 g of rubradirin in 100 ml of glacial acetic acid is added 30ml of water and the resulting mixture is stirred for 20 days at roomtemperature. A precipitate (compound V) is isolated by filtration,washed with water and dried; yield, 2.75 g. This compound assays 150BU/mg vs. S. lutea. Anal. Calcd. for C₄₀ H₃₃ N₃ O₁₆ : C, 59.18; H, 4.10;N, 5.18; MW 811.68. Found: C, 58.27; H, 4.31; N, 4.97; m/e 811.

The filtrate is evaporated in a nitrogen stream. The resulting residueis dissolved in chloroform and this solution is decolorized byextraction with a 3% aqueous sodium carbonate solution. The driedchloroform solution is diluted by addition of 1/5 volume of methanol.This, when partially evaporated, affords a total of 84 mg of whitecrystals of compound VI in two crops, m.p. 150°-153°. The infraredspectrum of this material shows significant absorbances at 1550, and1360-1380 cm⁻.

Anal. Calcd. for C₈ H₁₅ NO₅ : C, 46.82; H, 7.37; N, 6.83. Found: C,46.70; H, 7.63; N, 6.67.

EXAMPLE 3--Degradation Of Compound V

A saturated solution of ammonia and methanol, containing 700 mg ofcompound V is stirred 16 hours at room temperature, then evaporated todryness on a rotary evaporator. The residue is triturated with a 2-phasemixture of 200 ml of chloroform and 100 ml of 0.1 N sulfuric acid. Thisis then filtered, affording a precipitate containing compound VII and afiltrate. The chloroform phase of the filtrate, when washed, evaporated,and dried, yields 280 mg of compound III.

Compound VII is purified from the first precipitate by crystallizationfrom hot methanol; yield, 300 mg.

Anal. Calcd. for C₁₇ H₁₃ N₃ O₈ : m/e 387. Theory by high resolution MS387.0702. Found: 387.0702.

EXAMPLE 4--Base Degradation Of Rubradirin B: Preparation Of Compound XAnd Compound XI

A suspension of 1 g of rubradirin B in 75 ml of water is brought to pH11 by addition of 1 N sodium hydroxide solution over the period of 1hour. After 16 hours the pH is 7.5 and it is then raised and kept at pH12 for two days by addition of 1 N sodium hydroxide. The solution isacidified to pH 2 with 6 N sulfuric acid and all of the insoluble, andchloroform-soluble fractions are pooled; yield, 700 mg. This material ischromatographed on 100 g of silica gel 60, buffered at pH 5.8 (54.4 g ofKH₂ PO₄ /Kg of SiO₂) in a column of 3.3 cm diameter. This is eluted with850 ml of chloroform and these eluates (fractions 1-10) are discarded.Choroform:methanol, 95:5 v/v is the next eluant and 10-ml fractions arecollected (fractions 11-127). Fractions 35-46 are pooled on the basis oftlc (thin layer chromatography) analysis in the system: pH 5.8-bufferedsilica gel, HF 254: chloroform:methanol (98:2). It displays an R_(f) of0.22 vs. 0.32 for the corresponding compound lll from rubradirin and0.66 for starting material rubradirin B.

The product, compound X (180 mg), which subsequently crystallizes fromdeutero-chloroform, assays at 6 BU/mg (S. lutea). [α]_(D) =+108° (c,0.02, acetone). Anal. Calcd. for C₂₃ H₂₃ N₁ O₈ : m/e 441, Theory441.1424. Found: 441.1413.

Compound XI is eluted from the above column with methanol and isolatedby evaporation.

EXAMPLE 5--Degradation of Rubradirin In Acetic Anhydride And Pyridine:Preparation Of Compound VIII

A mixture of 10 g of rubradirin, 125 ml of pyridine, amd 25 g of aceticanhydride is refluxed for 4 hours. It is then poured on ice and theresulting precipitate is removed by filtration and recrystallized frommethanol twice to give 300 mg of white crystals of compound VIII, mp212°-213°.

Anal. Calcd. for C₁₂ H₉ NO₄ : C, 62.34; H, 3.92; N, 6.06. Found: C,62.49; H, 3.78; N, 6.05.

EXAMPLE 6

By substituting rubradirin B, or compound IV, or compound V, or compoundVII, or compound XI, for rubradirin in Example 5, there is obtainedcompound VIII.

Rubradirin B can be prepared as follows:

A. Fermentation

An agar slant of Streptomyces achromogenes var. rubradiris, NRRL 3061*,is used to inoculate a series of 500-ml Erlenmeyer flasks eachcontaining 100 ml of sterile seed medium consisting of the followingingredients:

Glucose monohydrate--25 g/liter

Pharmamedia **--40 g/liter

Tap water q.s.--1 liter

The flasks are incubated for 3 days at 28° C. on a Gump rotary shakeroperating at 250 r.p.m.

Seed inoculum (5%), prepared as described above, is used to inoculate aseries of 500-ml Erlenmeyer flasks each containing 100 ml of sterilefermentation medium consisting of the following ingredients:

Starch--10 g/liter

Corn steep liquor--20 g/liter

Distillers' solubles--15 g/liter

Mg (NO₃)₂.6H₂ O--3.8 g/liter

Tap water q.s.--1 liter

The fermentation medium presterilization pH is 7.2.

The fermentation flasks are incubated at 28° C. on a Gump rotary shakeroperating at 250 r.p.m. The fermentation flasks are harvested afterabout 3 to 4 days. A typical shake flask fermentation is depicted below.The assay is against the microorganism S. lutea. It is a disc plateassay using 0.1 M phosphate buffer, pH 7.85, as diluent.

    ______________________________________                                        Day               Assay, Biounit/ml                                           ______________________________________                                        1                 trace                                                       2                 104                                                         3                 160                                                         4                  64                                                         ______________________________________                                         NOTE:-                                                                        One Biounit corresponds to the dilution factor of the sample to yield an      inhibition zone of 20 mm.                                                

B. Recovery

Whole broth from a fermentation, as described above, is slurried with 4percent of its weight of diatomaceous earth and filtered. The filtercake is washed with 1/10 volume of water and the wash is added to theclear beer. The clear beer is adjusted to pH 4.0 with 6 N sulfuric acidand filtered with the aid of diatomaceous earth. The spent beer isdiscarded. The wet cake is leached with ethyl acetate and the solventphase is then concentrated to an aqueous phase. The latter isfreeze-dried. The residue is dissolved in ethyl acetate and diluted with4 volumes of Skellysolve B. The precipitate which is collected and driedcontains a mixture including rubradirin and rubradirin B.

C. Purification

A one gram quantity of crude preparation containing rubradirin B,prepared as described above, is chromatographed on 500 g of silica gel G(70-230 mesh, E. Merck), buffered at pH 5.8. The first elution with 1500ml of chloroform is discarded. Thereafter 20 ml fractions are collected.Tubes 201 to 470 contain rubradirin by tlc. The elution solvent ischanged to chloroform:methanol (97:3). Tubes 471-510 contain a mixtureof rubradirin and rubradirin B. The solids in this fraction are isolatedby concentration and precipitation is Skellysolve B, 310 mg.

The combined solids from the above chromatography and two similar ones,660 mg total, are then dissolved and suspended in 30 ml of chloroform,and this is stirred for 1 hour and filtered. The semicrystallineprecipitate, 160 mg, is found to be essentially pure rubradirin B bytlc.

The tlc is run on Eastman silica gel (#6060) sheets with the solventsystem ethyl acetate-acetone-water (8:5:1) and bioautographed on traysseeded with S. lutea. Approximately 0.5 γ of line product preparationsand correspondingly lesser amounts of higher purity preparations areapplied for analyses.

Preparations are assayed after they have been adjusted to pH 3.0 anddried in vacuum. Dilutions are made in methanol and a quantity of 0.08ml is applied to 12.7 mm assay discs which are dried and placed on agartrays seeded with S. lutea. Assays are expressed as biounlts.

Salts of the compounds of the subject invention, except compound VI, areformed employing the free acid and an inorganic or organic base. Thesalts can be prepared as for example by suspending the free acid inwater, adding a dilute base until the pH of the mixture is about 7 to 8,and freeze-drying the mixture to provide a dried residue consisting ofthe salt. Salts which can be formed include the sodium, potassium, andcalcium. Other salts including those with organic bases such as primary,secondary, and tertiary mono-, di-, and polyamines can also be formedusing the above-described or other commonly employed procedures. Thesalts can be used for the same purposes as the parent free acid.

The compounds of the subject invention can be acylated under standardacylating conditions with an appropriate acid halide or anhydride togive the acylated compounds. For example, compounds III and X can beacylated at positions 4, 5 and 11 to give the triacylate of saidcompounds. Compound V can be acylated at positions 4 and 11 of the Ansamoiety and at the free hydroxyl at position 3 of the nitro-sugar moiety.Compound VI can be acylated on the anomeric carbon. Compounds IV and XIcan be esterified with diazomethane or lower alcohols (1 to 4 carbonatoms, inclusive) under acid catalysis to form esters. The acylation iscarried out in the presence of an acid-binding agent. Suitableacid-binding agents include: amines such as pyridine, quinoline, andisoquinoline, and buffer salts such as sodium acetate. The preferredbase is pyridine. Carboxylic acids suitable for acylation include (a)saturated or unsaturated, straight or branched chain aliphaticcarboxylic acids, for example, acetic, propionic, butyric, isobutyric,tertbutylacetic, valeric, isovaleric, caproic, caprylic, decanoic,dodecanoic, lauric, tridecanoic, myristic, pentadecanoic, palmitic,margaric, stearic, acrylic, crotonic, undecylenic, oleic, hexynoic,heptynoic, octynoic acids, and the like; (b) saturated or unsaturated,alicyclic carboxylic acids, for example, cyclobutanecarboxylic acid,cyclopentanecarboxylic acid, cyclopentenecarboxylic acid,methylcyclopentenecarboxylic acid, cyclohexanecarboxylic acid,dimethylcyclohexanecarboxylic acid, dipropylcyclohexanecarboxylic acid,and the like; (c) saturated or unsaturated, alicyclic aliphaticcarboxylic acids, for example, cyclopentaneacetic acid,cyclopentanepropionic acid, cyclohexaneacetic acid, cyclohexanebutyricacid, methylcyclohexaneacetic acid, and the like; (d) aromaticcarboxylic acids, for example, benzoic acid, toluic acid, naphthoicacid, ethylbenzoic acld, isobutylbenzoic acid, methylbutylbenzoic acid,and the like; and (e) aromatic aliphatic carboxylic acids, for examplephenylacetic acid, phenylpropionic acid, phenylvaleric acid, cinnamicacid, phenylpropiolic acid, and naphthylacetic acid, and the like. Also,suitable halo-, nitro-, hydroxy-, amino-, cyano-, thiocyano-, and loweralkoxy-hydrocarbon carboxylic acids include hydrocarbon carboxylic acidsas given above which are substituted by one or more of halogen, nitro,hydroxy, amino, cyano, or thiocyano, or lower alkoxy, advantageouslylower alkoxy of not more than six carbon atoms, for example, methoxy,ethoxy, propoxy, butoxy, amyloxy, hexyloxy groups and isomeric formsthereof. Examples of such substituted hydrocarbon carboxylic acids are:

mono-, di- and trichloroacetic acid;

α- and β-chloropropionic acid;

α- and γ-bromobutyric acid;

α- and δ-iodovaleric acid;

mevalonic acid;

2- and 4-chlorocyclohexanecarboxylic acid;

shikimic acid;

2-nitro-1-methylcyclobutanecarboxylic acid;

1,2,3,4,5,6-hexachlorocyclohexanecarboxylic acid;

3-bromo-2-methylcyclohexanecarboxylic acid;

4- and 5-bromo-2-methylcyclohexanecarboxylic acid;

5- and 6-bromo-2-methylcyclohexanecarboxylic acid;

2,3-dibromo-2-methylcyclohexanecarboxylic acid;

2,5-dibromo-2-methylcyclohexanecarboxylic acid;

4,5-dibromo-2-methylcyclohexanecarboxylic acid;

5,6-dibromo-2-methylcyclohexanecarboxylic acid;

3-bromo-3-methylcyclohexanecarboxylic acid;

6-bromo-3-methylcyclohexanecarboxylic acid;

1,6-dibromo-3-methylcyclohexanecarboxylic acid;

2-bromo-4-methylcyclohexanecarboxylic acid;

1,2-dibromo-4-methylcyclohexanecarboxylic acid;

3-bromo-2,2,3-trimethylcyclopentanecarboxylic acid;

1-bromo-3,5-dimethylcyclohexanecarboxylic acid;

homogentisic acid, o-, m-, and p-chlorobenzoic acid;

anisic acid;

salicyclic acid;

p-hydroxybenzoic acid;

β-resorcyclic acid;

gallic acid;

veratric acid;

trimethoxybenzoic acid;

trimethoxycinnamic acid;

4,4'-dichlorobenzilic acid;

o-, m-, and p-nitrobenzoic acid;

cyanoacetic acid;

3,4- and 3,5-dinitrobenzoic acid;

2,4,6-trinitrobenzoic acid;

thiocyanoacetic acid;

cyanopropionic acid;

lactic acid;

ethoxyformic acid (ethyl hydrogen carbonate);

and the like.

The acylated compounds, amides and esters, as described herein, can beused for the same purposes as disclosed for the parent compounds.Additionally, the acylates can be used to upgrade the parent compound,i.e. the parent compound is acylated, then deacylated under standardconditions to give a higher purity parent compound.

EXAMPLE 7--Ethylamide Of Compound IV

A solution containing 700 mg of rubradirin in 20 ml of 30% aqueousethylamine is stored at room temperature for 36 hours, then evaporatedto dryness on a rotary evaporator. The residue is redissolved in 20 mlof water, brought to pH 1.5 with 2N HCl, and extracted with a total of40 ml of chloroform in several batches. The extract is concentrated to aresidue (520 mg) which is chromatographed over 125 g of pH 5.8-bufferedsilica gel in a 3 cm (dia.) column developed by chloroform: methanol(98:2). The residue from fractions (20 ml) 50-70, 110 mg, if identifiedas rubransarol A. The residue from fractions 41-45 (200 mg) iscrystallized from a chloroform and butanone mixture to give the titlecompound: 170 mg, M. 209° dec. Anal. Cacd. for C₂₇ H₃₀ N₄ O₁₂ : C,53.82; H, 5.02; N, 9.30. Found: C, 53.05; H, 5.11; N, 9.13.

EXAMPLE 8--Methylamide Of Compound IV

A 7 g quantity of rubradirin, dissolved in 100 ml of 40% aqueousmethylamine, is stored at room temperature for 20 hours. It is thenconcentrated to 25 ml on a rotary evaporator and brought to pH 2 with 2Nhydrochloric acid. The precipitate, after collection and drying, issuspended in methyl ethyl ketone affording pale yellow crystals of thetitle compound: 3.7 g (94%), M. 265° dec. This material has twotitratable groups, PK_(a).5.3 and 7.8 in 85% aqueous dimethyl sulfoxide.Anal. Calcd. for C₂₆ H₂₈ N₄ O₁₂ : C, 53.06; H, 4.80; N, 9.52; M.W. 588.Found: C, 53.08; H, 4.88; N, 9.34; M⁺ 588.

EXAMPLE 9--Ethylamide Of Compound XI

Rubradirin B (1 g, 1.25 mmol) is dissolved in 20 ml of 30% aqueousethylamine and stored 16 hours at room temperature. It is thenevaporated to a solid residue on a rotary evaporator and to this isadded a mixture of 20 ml of 0.2 N HCl, 20 ml of chloroform, and 20 ml ofacetone. Following filtration, the precipitate is washed thoroughly withacetone, leaving 450 mg of yellow solid. Recrystallization from boilingdioxane afforded 170 mg of pale yellow crystals of the title compound:M. >285° . Anal. Calcd. for C₁₉ H₁₇ N₃ O₇ : C, 57.14; H, 4.29; N, 10.52;M.W. (High resolution mass spectroscopy) 399.10664. Found: C, 56.84; H,4.34; N, 10.49; M.W., 399.10733.

I claim:
 1. A process for preparing the compound of the followingstructural formula: ##STR9## which comprises reacting the antibioticrubradirin with pyridine and acetic anhydride at reflux.
 2. A processfor preparing the compound of the following structural formula:##STR10## which comprises reacting a compound selected from the groupconsisting of rubradirin B, compound IV, compound V, compound VII andcompound XI with pyridine and acetic anhydride at reflux.
 3. A processfor preparing the compound of the following structural formula:##STR11## which comprises reacting a compound selected from the groupconsisting of rubradirin, rubradirin B, compound IV, compound V,compound VII and compound XI with pyridine and acetic anhydride atreflux.